PROJECT SUMMARY Some of the most important human pathogens includes those that cause difficult to treat infections due to their resistance to commonly used antibiotics. For Gram negative organisms, the main resistance mechanism includes an impermeable cell envelope in conjunction with activities of expulsion (efflux) pumps. This group includes Pseudomonas aeruginosa, a pathogen of immunocompromised individuals and those with cystic fibrosis, with numerous efflux pumps encoded in its genome. This proposal aims at creating new enabling tools for drug discovery, by exploring the feasibility of using periplasmically expressed single domain antibodies (nanobodies) capable of blocking biological processes that take place in this compartment. The work proposed here will target the periplasmic components of the MexXY-OprM efflux pump, with the objective of identifying nanobodies that interfere with antibiotic efflux. In the first Aim, recombinant MexX and the fused periplasmic domains of MexY will be purified and these will be used to select binders from a synthetic library of nanobodies displayed on yeast surface. These will be transferred to bacterial expression vectors and introduced into P. aeruginosa where the secretion of antibodies into the periplasm should interfere with the assembly or function of MexXY-OprM, rendering the bacteria sensitive to antibiotics that are substrates of this efflux pump. A number of genetic and biochemical approaches will be used to map the nanobody epitopes on the Mex proteins. Nanobodies showing weak affinities will be subjected to several round of affinity maturation. The project should generate not only information about functional domains of an efflux pump but also tools suitable for screening and validation of small molecule inhibitors of antibiotic efflux.